PROJECT SUMMARY. RNA interference (RNAi), a process in which small interfering (siRNA) molecules inhibit gene expression, is of broad therapeutic potential in the treatment of a variety of diseases but is presently hampered by delivery challenges including susceptibility of siRNA degradation, rapid clearance, and cell impermeability. Local delivery circumvents some of these challenges, but siRNA must still be protected from degradation and effectively enter target cells within the tissue of interest. Thus, there remains a significant unmet need for platform delivery technologies capable of controlled and effective in vivo delivery of siRNA. LayerBio proposes to develop new delivery approach for siRNA therapeutics based on its proprietary LayerForm? technology. LayerForm is a coating process based on a layer-by-layer (LbL) nanofabrication technique that stably incorporates nucleic acid therapeutics from water into a thin polymer coating atop a surface. The polymer coating is engineered to erode at physiologically relevant time scales delivering siRNA or other drugs to target tissue. LayerForm offers the following benefits: (i) precise control of siRNA dosing and release, (ii) effective transfection of nucleic acids, (iii) fully biocompatible materials, (iv) modular platform capable of delivery of a variety of nucleic acids, and (v) facile processing. The use of siRNA-based strategies in regenerative medicine and tissue engineering is a promising application of RNA interference where effective therapeutics to promote wound healing would offer a significant health benefit. Typical wounds would require local delivery of siRNA where the LayerForm delivery technology is well suited. LayerBio has developed siRNA incorporated LbL coatings atop wound dressings to accelerate the healing of chronic wounds. An siRNA oligonucleotide has been selected for modulation of key targets that coordinate a cascade of events that occur during normal wound healing, but are impaired in chronic wounds. During in vivo feasibility studies, LayerForm siRNA-LbL coated dressings demonstrated accelerated wound healing in diabetic mice. This Phase 1 project will further advance its LayerBio's technology platform toward clinical translation by demonstrating feasibility for process scale-up. Further, current siRNA LbL formulations are fabricated atop non-degradable nylon mesh that must be removed from the wounds. LayerBio will fabricate siRNA-LbL films atop suitable bioresorbable dressing materials capable of being absorbed into the wound. These advancements will be validated in an in vivo study for efficacy in a diabetic mouse model of wound healing. Results of this Phase 1 effort will form the basis for Phase 2 optimization, process scale-up, and toxicology testing. Success in this project will also motivate the expansion of LayerBio's technology platform for delivery of other nucleic acid therapeutics and other disease indications.